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| United States Patent |
5,759,848
|
|
Nagoshi
, et al.
|
June 2, 1998
|
Biological indicator
Abstract
Described is a biological indicator which comprises an indicator
microorganism and a film which allows the permeation of a sterilizing gas
but does not allow the permeation of the microorganism, said microorganism
having been wrapped with the film.
| Inventors:
|
Nagoshi; Jinko (Tonami, JP);
Ueda; Yuzo (Osawano-machi, JP);
Eidai; Naotake (Toyama, JP);
Wakabayashi; Yukio (Fuchu-machi, JP);
Nogami; Toshihiro (Toyama, JP)
|
| Assignee:
|
Fujiyakuhin Co., Ltd. (Oomiya, JP)
|
| Appl. No.:
|
796750 |
| Filed:
|
February 6, 1997 |
Foreign Application Priority Data
| Current U.S. Class: |
435/287.1; 435/287.4; 435/287.6; 435/287.7 |
| Intern'l Class: |
C12M 003/00 |
| Field of Search: |
435/287.1,287.4,287.6,287.7
|
References Cited
U.S. Patent Documents
| 4416984 | Nov., 1983 | Wheeler | 435/287.
|
| 4528268 | Jul., 1985 | Andersen et al. | 435/287.
|
| 4743537 | May., 1988 | McCormick et al. | 435/287.
|
| 5340741 | Aug., 1994 | Lemonnier | 435/288.
|
Primary Examiner: Redding; David A.
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier & Neustadt, P.C.
Claims
What is claimed is:
1. A biological indicator, which consists essentially of an indicator
microorganism and a film which allows the permeation of a sterilizing gas
but does not allow the permeation of the microorganism, said microorganism
having been substantially wrapped with the film alone, and wherein the
film has pores of 0.01 .mu.m to 0.5 .mu.m in pore size and wherein the
film has gas permeability of at least 1000 ml/cm.sup.2 /min/bar.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a biological indicator used for the verification
of the effects of disinfection, fumigation or sterilization of
microorganisms by using high pressure steam, a sterilizing gas, hydrogen
peroxide, ozone, electron beam, gamma rays or the like.
2. Description of the Related Art
Microorganisms are generally classified into two groups, that is, bacteria
and fungi and each group has a fixed shape and size. These microorganisms
exhibit pathogenicity when mixed in a pharmaceutical, cosmetics or food,
thereby exerting serious influences on the quality of the product, for
example, deterioration in its quality or impairment of its flavor. When
microorganisms or their dead bodies are mixed in an injection, on the
other hand, they have serious influences on the patient to whom the
injection has been administered, for example, by causing infectious
diseases.
With a view to maintaining the quality of a pharmaceutical, it is therefore
very important to remove such microorganisms from its production
environment and to take measures against the contamination of the
microorganisms into it. For the above purposes, it is inevitable to
perform disinfection, fumigation or sterilization of the production
environment, production equipment, materials, working clothes and the
like. In addition, in the medical job site, it is also inevitable to
perform disinfection, fumigation or sterilization of an operating room,
tools and materials for operation, an operating gown and operating clothes
in order to prevent hospital infection, thereby maintaining the medical
safety.
To verify that the disinfection, fumigation or sterilization work has
accomplished its object, it is the common practice to investigate the
dying degree of the microorganisms, whether the disinfection, fumigation
or sterilization has been performed with certainty, using living
microorganisms as an indicator.
Examples of the microorganisms used generally for a biological indicator
include thermophilic bacteria, chemical-resistant bacteria,
poisonous-gas-resistant bacteria and radiation-resistant bacteria, more
specifically, Bacillus subtilis var. niger, Bacillus stearothermophilus or
Bacillus pumilus. As the biological indicator, a carrier onto which some
of these microorganisms have been adhered in a viable count of 10.sup.2
-10.sup.8 is used. Although such active microorganisms are widely used as
a biological indicator (BI), there is a danger that owing to incomplete
wrapping, they leak from the wrapping or pass through the wrapping,
scatter around and contrary to the original purpose, become a cause of
bacterial pollution. It is the present situation that particularly in the
case where containers, clothes or the like are sterilized by using a
poisonous gas, hydrogen peroxide or ozone or where a production site of
pharmaceuticals, an operating room or the like is sterilized by
fumigation, the effects of the above-described biological indicator cannot
be verified without using an excess amount of a poisonous gas, hydrogen
peroxide or ozone, because the wrapping for tightly sealing of the
above-described biological indicator markedly prevents the gas
permeability. An excess amount of the poisonous gas, hydrogen peroxide or
ozone increases not only an adverse effect on the human body, environment,
equipment and the like but also a danger of causing a fire. Accordingly,
there is a strong demand for the establishment of a method for carrying
out disinfection, fumigation or sterilization at more appropriate
concentration of the above-described sterilizing gas.
SUMMARY OF THE INVENTION
An object of the present invention is therefore to provide a biological
indicator which makes it possible to verify the sterilization condition
effectively and safely without using an excess amount of a sterilizing
gas.
With a view to attaining the above object, the present inventors have
carried out an extensive investigation. As a result, it has been found
that by employing, as a wrapping material of indicator microorganisms, a
specific film which permits the permeation of a gas but does not permit
the permeation of the microorganisms, the microorganisms do not leak and
scatter from the film, while the sterilizing gas sterilizes the indicator
microorganisms at the same concentration as in the outside of the film.
Thus, the sterilization condition can be verified safely and effectively,
leading to the completion of the present invention.
The present invention, that is to say, provides a characteristic biological
indicator which comprises an indicator microorganism wrapped in a specific
film permitting the permeation of a sterilizing gas without permitting the
permeation of the microorganism.
Accordingly, the present invention makes it possible to provide a
biological indicator which allows the effective permeation of ozone,
hydrogen peroxide gas or poisonous gas and causes neither leakage nor
permeation of the microorganisms from the wrapping film and which is used
for the confirmation of the effects of disinfection, fumigation or
sterilization. As a result, the concentration of ozone, hydrogen peroxide
gas or poisonous gas can be suppressed to the minimum, whereby
sterilization can be verified effectively and safely without scattering
the microorganisms around the place to be verified.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
No other particular limitation than the permeability of a sterilizing gas
and no permeability of microorganism, is imposed on the film to be used
for the biological indicator of the present invention insofar. Those
having pores through which not microorganisms but a sterilizing gas is
permeable are preferred. It is preferred that the pore size of the film is
0.01 .mu.m to 0.5 .mu.m, with 0.1 .mu.m to 0.3 .mu.m being particularly
preferred. In consideration of the necessity of the effective permeation
of a sterilizing gas, it is preferred that the gas permeability of the
film is at least 1000 ml/cm.sup.2 /bar, with at least 2000 ml/cm.sup.2
/bar being particularly preferred. Furthermore, from the viewpoint of the
gas permeability, the porosity of the film is preferably 50% or higher,
with 70% or higher being particularly preferred.
Incidentally, the gas permeability can be determined by calculating the gas
permeable amount per hour and per area after measuring the amount of the
gas permeated through a film under a fixed pressure. The porosity, on the
other hand, can be determined in accordance with the following equation by
using the difference between the weights of the film before and after
impregnation with a liquid.
##EQU1##
wherein: W.sub.1 : weight of the film
W.sub.2 : weight of the film after impregnated with a liquid
V: volume of the film, and
C: specific gravity of the liquid
No particular limitation is imposed on the material of the film insofar
other than heat-, chemical- and radiation-resistance without a decrease of
the killing effects of ozone, hydrogen peroxide or poisonous gas to
microorganisms. Examples of the material include cellulose ester,
cellulose acetate, cellulose nitrate, polyamide, polyvinylidene fluoride,
polytetrafluoroethylene, polycarbonate, polyethylene, nylon and
polypropylene.
The biological indicator according to the present invention can be obtained
by having an indicator microorganism borne on a carrier and then packing
it with the above-described film. Here, examples of the usable indicator
microorganism generally include thermophilic bacteria, chemical-resistant
bacteria, poisonous-gas-resistant bacteria and radiation-resistant
bacteria, for example, Bacillus subtilis var. niger, Bacillus
stearothermophilus or bacillus pumilus. As the carrier, a filter paper is
generally employed. Porous synthetic resins and ceramics can also be used.
The viable count of the microorganisms which have been borne on the
carrier may be 10.sup.2 -10.sup.8.
For the purpose of transportation or preservation, the biological indicator
according to the present invention may be contained in a container or the
like which does not allow the permeation of both a gas and microorganisms.
The verification of the sterilizing effects using the biological indicator
according to the present invention is carried out in a conventional
manner. Described specifically, the biological indicator of the present
invention is positioned on the place to be sterilized and sterilization is
effected using an ozone gas, ethylene oxide gas, hydrogen peroxide gas or
formalin gas. The indicator microorganism is then taken out from the
wrapping and incubated on a nutrient medium, followed by the observation
of the presence or absence of the proliferation of the microorganisms.
The present invention will hereinafter be described in more specifically by
the following examples. It should however be borne in mind that the
present invention will not be limited to or by the following examples.
EXAMPLE 1
A filter paper to which Bacillus subtilis var. niger had been adhered in a
viable count of 10.sup.6 as an indicator microorganism was wrapped with a
polyvinylidene fluoride film having a pore size of 0.22 .mu.m, gas
permeability of 4000 ml/cm.sup.2 /min/bar and porosity of 75% by the heat
sealing method, whereby a biological indicator was prepared.
Test 1
Each of the biological indicator obtained in Example 1, a biological
indicator (S) which was Example 1 without wrapping, and two commercially
available biological indicators (A) and (B) to which Bacillus subtilis
var. niger had been adhered in a viable count of 10.sup.6, was exposed to
an ozone gas and ethylene oxide gas respectively under the conditions
described below. Thereafter, each of the indicators was taken out from the
wrapping except the indicator (S) and the sterilization effects were
confirmed using a soybean-casein-digest agar (SCD) medium.
The results are shown in Tables 1 and 2.
TABLE 1
______________________________________
Exposure Test to Ozone
Example 1
(S) (A) (B)
______________________________________
300 ppm Positive Positive Positive
Positive
500 ppm Negative Negative Positive
Positive
700 ppm Negative Negative Positive
Positive
900 ppm Negative Negative Positive
Negative
1200 ppm Negative Negative Negative
Negative
______________________________________
Exposure conditions: at 20.degree. C. for 120 min.
TABLE 2
______________________________________
Exposure Test to Ethylene Oxide Gas
Example 1
(S) (A) (B)
______________________________________
30 min Positive Positive Positive
Positive
60 min Negative Negative Positive
Positive
90 min Negative Negative Positive
Positive
120 min Negative Negative Negative
Negative
______________________________________
Exposure conditions: at 60.degree. C., 75%, 640 mg/l
Test 2
Each of the biological indicator obtained in Example 1, a biological
indicator (S) which was Example 1 without wrapping, and two commercially
available biological indicators (A) and (B) to which Bacillus subtilis
var. niger had been adhered in a viable count of 10.sup.6 were subjected
to an incubation test on an SCD medium without being taken out from the
wrapping except the indicator (B).
The results are shown in Table 3.
TABLE 3
______________________________________
Leakage and Permeation Test
Example 1
(S) (A) (B)
______________________________________
Results Negative Positive Positive
Positive
______________________________________
It has been confirmed that the biological indicator of Example 1 has good
gas permeability and causes neither leakage nor permeation of the
microorganisms from the wrapping.
EXAMPLE 2
A filter paper to which Bacillus subtilis var. niger had been adhered in a
viable count of 10.sup.6 as an indicator microorganism was wrapped with a
polytetrafluoroethylene film having a pore size of 0.2 .mu.m, gas
permeability of 4000 ml/cm.sup.2 /min/bar and porosity of 85% by the heat
sealing method, whereby a biological indicator was prepared.
Test 3
Each of the biological indicator obtained in Example 2, a biological
indicator (S) which was Example 2 without wrapping, and two commercially
available biological indicators (A) and (B) to which Bacillus subtilis
var. niger had been adhered in a viable count of 10.sup.6, were exposed to
a hydrogen peroxide gas and formalin gas, respectively, under the
conditions described below. Thereafter, each of the indicators was taken
out from the wrapping except the indicator (S) and the sterilization
effects were confirmed using a soybean-casein-digest agar (SCD) medium.
The results are shown in Tables 4 and 5.
TABLE 4
______________________________________
Exposure Test to Hydrogen Peroxide Gas
Example 2
(S) (A) (B)
______________________________________
500 ppm Positive Positive Positive
Positive
1000 ppm Negative Negative Positive
Positive
1500 ppm Negative Negative Positive
Positive
2000 ppm Negative Negative Positive
Negative
3200 ppm Negative Negative Negative
Negative
______________________________________
Exposure conditions: at 20.degree. C. for 120 min.
TABLE 5
______________________________________
Exposure Test to Formalin Gas
Example 2
(S) (A) (B)
______________________________________
1000 ppm Positive Positive Positive
Positive
2000 ppm Negative Negative Positive
Positive
3000 ppm Negative Negative Positive
Positive
4000 ppm Negative Negative Negative
Negative
______________________________________
Exposure conditions: at 20.degree. C., 70%
Test 4
Each of the biological indicator obtained in Example 2, a biological
indicator (S) which was Example 2 without wrapping, and two commercially
available biological indicators (A) and (B) to which Bacillus subtilis
var. niger had been adhered in a viable count of 10.sup.6, was subjected
to an incubation test on an SCD medium without being taken out from the
wrapping.
The results are shown in Table 6.
TABLE 6
______________________________________
Leakage and Permeation Test
Example 2
(S) (A) (B)
______________________________________
Results Negative Positive Positive
Positive
______________________________________
It has been confirmed that the biological indicator of Example 2 has good
gas permeability and causes neither leakage nor permeation of the
microorganisms from the wrapping.
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